Because most human infections affect or gain entry through the mucosal surfaces of the oro-gastrointestinal, respiratory, and genitourinary tracts, future vaccines to elicit protection at these sites will likely be administered by mucosal routes, intragastrically (i.g.) or intranasally (i.n.). Among numerous strategies designed to enhance responses to mucosal vaccines, bacterial heat-labile enterotoxins such as cholera toxin (CT) and the labile toxins of Escherichia coli (LT-I, LT-IIa and LT-IIb) have been exploited as both powerful immunostimulatory adjuvants and carrier molecules coupled to vaccine antigens. However, it is not known precisely which tissues and cells are targeted by these adjuvants and immunogens, and how and where they are processed and presented to T cells to initiate the immune response. Some antigens when given i.g. alone or coupled to the B subunit of CT suppress immune responses in the phenomenon known as "oral tolerance". The mechanisms by which these two divergent responses are induced are poorly understood, but are critical in the design and development of mucosal vaccines, whether intended for generating protection against infections, or for suppressing undesirable immune responses such as in autoimmune diseases or allergies. The precise tissue sites and characteristics of the cells involved in the uptake of vaccine antigens and adjuvants, as well as the mode of action of the adjuvants in activating antigen-presenting cells, are critical in determining the nature of the ensuing immune response. Limitations in previous technologies for tracking the uptake of antigens and adjuvants in vivo can be overcome by applying the emerging novel ImageStream (Amnis Corp.) technology. In this collaborative project between experts in mucosal immunology and flow and imaging cytometry, the uptake of fluorochrome-labeled mucosal immunogens based on antigen-enterotoxin chimeric proteins, and of enterotoxin adjuvants (or their nontoxic derivatives), will be studied in mice. The Specific Aims are: (1) to determine the cellular pathways of uptake of antigen-enterotoxin chimeric proteins administered i.g. or i.n., and to characterize the type, location, and activation of antigen-presenting cells involved;and (2) to compare the cellular pathways of uptake of the intact enterotoxin adjuvants, their binding- site mutants, and B subunits, in comparison with control proteins, when administered i.g. or i.n. The results are expected to identify the precise sites of uptake of these immunogens and adjuvants, define the characteristics of the cells stimulated by them, and thereby determine the type of immune responses that are induced. Such information will be important for the design of future mucosally applied vaccines. PUBLIC HEALTH RELEVANCE: New generations of vaccines administered by intranasal or intragastric routes are being developed to elicit protection at the mucosal surfaces where most human infections arise, and have the additional advantage of being needle-free. However, the precise tissues and cells that takes up and responds to mucosally administered vaccine antigens and adjuvants are not adequately understood, yet this knowledge is essential for the design of such vaccines whether intended for protection against infections or to suppress undesirable responses in autoimmune diseases or allergies. This proposal will apply novel imaging technology based on the Amnis Image Stream to identify the tissues and cells targeted by mucosal vaccines and adjuvants, and to characterize the antigen-presenting cells that initiate the immune responses to them.